Broad Spectrum Antiviral and Methods of Use

ABSTRACT

A method for the prevention or treatment of Influenza virus infection or Adenovirus infection by administering an effective amount of a compound of Formula (I), Formula (II), or similar compound to an individual in need is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of the filing ofU.S. Provisional Patent Application Ser. No. 60/792,738, entitled “BroadSpectrum Antiviral and Methods of Use”, filed on Apr. 17, 2006, and thespecification and claims thereof are incorporated herein by reference.

FIELD OF THE INVENTION

A method for the prevention or treatment of DNA viruses, RNA viruses,and DNA and RNA reverse transcribing viruses infection by administeringan effective amount of a compound of Formula (I), Formula (II) or aderivative thereof to an individual in need is provided.

BACKGROUND OF THE INVENTION

Viruses can be divided into several (arbitrary) classifications basedupon the type of nucleic acid that they carry and the mode ofexpression. Table 1 illustrates one classification scheme based upon“The International Code of Virus Classification and Nomenclature”, andthe classification system developed by Dr. David Baltimore (incorporatedherein by reference)

TABLE 1 Group Name Nucleic Acid Type Order Examples DNA viruses Doublestranded Order: Ex. Enterobacteria phage T4 DNA viruses CaudoviralesUnassigned Ex. Family Adenoviridae viruses Ex. Family Herpesviridae Ex.Family Polymaviridae (simiam virus 40) Ex. Family Poxviridae (Cowpoxvirus, Variola virus - smallpox) Single stranded Unassigned DNA virusesbacteriophages Unassigned Ex. Family Parvoviridae viruses RNA virusesDouble stranded Unassigned Ex. Family Reoviridae RNA viruses viruses (+)single-stranded Order: Ex. Family Coronaviridae (coronavirus, RNA ormRNA-like Nidovirales SARS) viruses Unassigned Ex. Family Flaviviridae(Yellow fever viruses virus, West Nile virus, Hepatitis C virus) Ex.Family Picornaviridae (poliovirus, rhino virus, hepatitis A virus) Ex.Togaviridae (Rubella virus) (−) single-stranded Order: Ex. FamilyParamyxoviridae (measles RNA viruses Mononegavirales virus, mumps virus)(non-segmented Ex. Family Rhabdoviridae (rabies virus) negative strandedviruses) Segmented Ex. Family Orthomyxoviridae (Influenza negativestranded viruses) viruses DNA and RNA Single-stranded Unassigned Ex.Family Retroviridae (Retroviruses, reverse RNA reverse viruses HIV)transcribing transcribing viruses viruses Double-stranded Unassigned Ex.Family Hepadnaviridae (Hepatitis B DNA reverse viruses virus)transcribing virus

The design and discovery of new antiviral drugs can be directed againsteither viral or cellular targets. Drugs that inhibit viral proteins aremore likely to be virus-specific and are more prone to the developmentof resistance. Thus, there is an urgent need for broad spectrumantiviral drugs that can be used in mono- or combined therapy to treatthe numerous viral diseases caused by DNA viruses, RNA viruses and DNAand RNA reverse transcribing viruses for which limited or no therapeuticoptions are currently available. The demand for new effective and safeantiviral strategies has become an increasingly important problem tosolve in recent years due to the rising prevalence of chronic viralinfections such as HIV and hepatitis B and C, the emergence of newviruses or viral strains such as the SARS coronavirus and pathogenicAvian Influenza strains, the ever-present threat of viral pandemics fromagents like virulent strains of influenza A, and the potential danger ofhemorrhagic fever viruses and eradicated viruses such as variola virusbeing exploited as bioterrorist weapons.

A limited number of chronic viral diseases, which affect millions ofpeople world-wide (e.g., 40 million individuals are HIV-infected), canbe controlled to some degree, but no cures are currently available. As aresult, infected individuals provide a reservoir for the respectivevirus through which naïve individuals become infected, thereby,perpetuating the problem of viral infection of significant humanpopulations with these pathogens. Currently available antiviral drugs,such as the nucleoside reverse transcriptase inhibitors (NRTIs) used toinhibit viral replication of specific pathogenic viruses, have resultedin recognizable improvements in the ability to control infections withthese pathogens and to improve the quality and length of life ofinfected individuals. A partial list of currently available antiviraldrugs used to inhibit viral replication of specific pathogenic virusesis provided in Appendix 2 (for eg. the NRTIs). The therapies listed haveresulted in recognizable improvements in the ability to controlinfections from these pathogens and to improve the quality and length oflife of infected individuals. However, currently available classes ofantiviral agents have limited utilities due to their narrow scope ofactivities against different viruses and/or problems with significantdrug-induced toxicities. In addition, the modes of action of the NRTIsand other drugs in current clinical use predispose to the development ofdrug resistance through viral mutations. Finally, many currentlyavailable drugs have considerable side effects that prevent their widespread use to achieve treatment or prevention goals.

SUMMARY OF THE INVENTION

There is a clear need for new antiviral agents and combination therapiesto suppress diseases caused by DNA viruses, RNA viruses and DNA and RNAreverse transcribing viruses. The compositions and methods of thepreferred embodiments provide such agents and associated methods oftreatment.

Amifostine is a pro-drug that is metabolized by alkaline phosphatase tothe reduced free thiol (WR-1065); oxidation of WR-1065 leads toformation of the disulfide (WR-33278). These reactions can be depictedschematically as follows:

Structural similarities can be observed between spermine (chemicalformula H₂N(CH₂)₃NH(CH₂)₂(CH₂)₂NH(CH₂)₃NH₂) and WR-1065 and WR-33278.Spermine is associated with nucleic acids and is thought to stabilizehelical structure, particularly in viruses. WR-1065 appears to functionas an analog of the polyamine spermine, and to compete with spermine forsites on DNA, and probably also on other nucleic acids and proteins.Thus, analogs of WR-1065 and the other compounds of preferredembodiments, as discussed below, may function as spermine or otherpolyamine analogs, and may mimic the antiviral and polyamine-likeactivity of WR-1065.

Thiol metabolites of amifostine are believed to be responsible for mostof the cytoprotective and radioprotective properties of amifostine.Amifostine is taken up by cells through a combination of passive andactive transport processes where it is metabolized to its active forms;these metabolites participate in a number of functions. These functionsinclude, but are not limited to, (i) protection against radiationinduced cytotoxicity and cell killing, (ii) protection againstradiation-induced mutagenesis/carcinogenesis, (iii) modulation oftopoisomerase I and topoisomerase II alpha activities, (iv) modulationof conformational changes in chromatid structure, (v) inhibition of cellcycle progression, (vi) inhibition of endonuclease activity, (vii)competition with spermine in polyamine transport systems, (viii)induction and repression of gene expression (effect dependent upon theparticular gene). Other activities include detoxifying cisplatin andother alkylating agents, scavenging free radicals, modulating apoptosis,and modifying the activity of specific enzymes/proteins.

To better understand their radioprotective activity, pharmacokineticstudies of amifostine and phosphonol have been performed; because of thestated goal of the studies, tissues known to be sensitive toradiation-induced damage were evaluated most extensively. The resultshave shown that the drugs are distributed to most normal tissues and toa lower degree to tumor tissue. The highest levels of drug distributionoccurred in the following tissues/organ systems (in order from highestto lowest): kidney, liver, salivary gland, heart, spleen, lung, muscle,and brain (bone marrow was also referred to as having high levels, butlevels relative to other tissues are not given) (Rasey et al. 1988,Pharmac Ther. 39: 33-43). It has further been demonstrated that certainnormal tissues are especially effective at uptake of the drugs (and/ortheir metabolites) and retention of those metabolites; these tissuesinclude: kidney, liver, salivary gland, and lung (Rasey et al., (1984),Radiat. Res. 97(3): 598-607). It is possible that other tissues thathave not been tested also perform like the above mentioned tissues.These studies establish the ability to obtain therapeutic levels ofthese drugs and/or their metabolites in the cells of these tissues/organsystems. Therapeutic drug levels are also expected to be obtained intissues or organs systems where drug pharmacokinetics resemble that ofthe above mentioned tissues.

Studies with amifostine incorporated into nanoparticles have shown thatthis preparation technique constitutes an effective mechanism fordelivering amifostine to multiple organ systems over a prolonged timeperiod. There was no significant difference in survival of bone marrowprogenitor cells in mice treated with 500 mg/kilogram amifostine byeither IP injection or by nanoparticles (administered by gavage,equivalent to oral dosing in humans) one hour prior to a dose of 8 to 9Gy whole-body gamma irradiation. These studies also showed significantprotection of jejunal crypt cells following the same exposures.Pharmacokinetic studies of amifostine distribution and retention in avariety of tissues following oral administration of PLGA/amifostinenanoparticles showed high levels of retention in all evaluated tissues30 minutes after administration. Retention of drug four hours afteradministration was demonstrated in the following tissues (in order fromhighest to lowest): liver, jejunum, stomach, ileum, duodenum, bonemarrow, and spleen.

The cytoprotective effects of amifostine appear to be dependent upon anumber of factors including, but not limited to, oxygen tension, pH,gene status (including the presence of a functional p53 gene), andenzyme status (including the expression of alkaline phosphatase in thecell membrane). Differences in these factors appear to be responsiblefor the differential cytoprotective effect mediated by amifostinebetween tumor cells and normal, nontumorigenic tissue.

According to one aspect amifostine, phosphonol and their derivatives andanalogs are particularly effective in inhibiting replication of DNAviruses, RNA viruses, and DNA and RNA reverse transcribing viruses.

Accordingly, in a first aspect a method of treating or preventing a DNAvirus, a RNA virus and/or a DNA or RNA reverse transcribing virusinfection in an individual in need thereof is provided, comprising thestep of administering to the individual an effective antiviral amount ofa compound or a pharmaceutically acceptable salt or solvate thereof,wherein the compound comprises Formula (I) or Formula (II)

or a pharmaceutically acceptable salt or solvate thereof, wherein X isselected from the group consisting of —PO₃H₂, hydrogen, acetyl,isobutyryl, pivaloyl, and benzoyl, wherein each of R₁, R₂, and R₃ isindependently selected from hydrogen and C₁₋₆ alkyl, and wherein n is aninteger of from 1 to 10.

In an embodiment of the first aspect, the compound is of Formula (I), R₁is methyl, R₂ is hydrogen, R₃ is hydrogen, and n is 3.

In an embodiment of the first aspect, the compound is of Formula (I), R₁is methyl, R₂ is hydrogen, R₃ is hydrogen, and X is hydrogen.

In an embodiment of the first aspect, the compound is of Formula (I), R₁is hydrogen, R₂ is hydrogen, R₃ is hydrogen, and n is 3.

In an embodiment of the first aspect, the compound is of Formula (I), R₁is hydrogen, R₂ is hydrogen, R₃ is hydrogen, and X is hydrogen.

In an embodiment of the first aspect, the compound is of Formula (II),R₁ is methyl, R₂ is hydrogen, R₃ is hydrogen, and n is 3.

In an embodiment of the first aspect, the compound is of Formula (II),R₁ is hydrogen, R₂ is hydrogen, R₃ is hydrogen, and n is 3.

In an embodiment of the first aspect, the compound is administered tothe individual at a daily dosage of from about 200 mg/m² to about 3000mg/m².

In an embodiment of the first aspect, the step of administering isselected from the group consisting of orally administering,subcutaneously administering, intravenously administering, parenterallyadministering, and administering by inhalation.

In an embodiment of the first aspect, the method further comprises thestep of administering to the individual an effective antiviral amount ofan antiviral drug, for example (see the list of antiviral drugs inAppendix 2) to treat or prevent an viral infection caused by a DNAvirus, a RNA virus, or a DNA or RNA reverse transcribing virus.

In a second aspect, a method of treating or preventing a human or animalviral infection caused by a DNA virus, a RNA virus, a DNA or a RNAreverse transcribing virus in an individual in need thereof is provided,comprising the step of administering to the individual an effectiveantiviral amount of amifostine, the free thiol form of amifostine, thedisulfide of amifostine (WR-33278), a combination of both of the freethiol and the disulfide of amifostine, or other structurally andfunctionally related compounds as described in this document.

In a third aspect, a method of treating or preventing an infectioncaused by a DNA virus, a RNA virus, or a DNA or RNA reverse transcribingvirus, or combination thereof in an individual in need thereof isprovided, comprising the step of administering to the individual aneffective antiviral amount of phosphonol, the free thiol form ofphosphonol, the disulfide of phosphonol, a combination of both the freethiol and the disulfide of phosphonol, or other structurally andfunctionally related compounds as described in this document.

In an embodiment of the second aspect, the viral infection is caused bya dsDNA virus.

In an embodiment of the third aspect, the viral infection is caused by adsDNA virus.

In an embodiment of the second aspect, the viral infection is caused bya dsRNA virus.

In an embodiment of the third aspect, the viral infection is caused by adsRNA virus.

In an embodiment of the second aspect, the viral infection is caused bya (+)ssRNA virus.

In an embodiment of the third aspect, the viral infection is caused by a(+)ssRNA virus.

In an embodiment of the second aspect, the viral infection is caused bya (−)ssRNA virus.

In an embodiment of the third aspect, the viral infection is caused by a(−)ssRNA virus.

In an embodiment of the second aspect, the viral infection is caused bya non Retroviridae ssRNA reverse transcribing virus.

In an embodiment of the third aspect, the viral infection is caused by anon Retroviradae ssRNA reverse transcribing virus.

In an embodiment of the second aspect, the viral infection is caused bya dsDNA reverse transcribing virus.

In an embodiment of the third aspect, the viral infection is caused by adsDNA reverse transcribing virus.

In an embodiment of the second aspect, the viral infection is caused bya ssDNA reverse transcribing virus.

In an embodiment of the third aspect, the viral infection is caused by assDNA reverse transcribing virus.

In a fourth aspect, a pharmaceutical kit is provided comprising apharmaceutical composition comprising a compound or pharmaceuticallyacceptable salt or solvate thereof in a pharmaceutically acceptablecarrier, the compound comprising Formula (I), Formula (II), or acombination thereof:

wherein X is selected from the group consisting of —PO₃H₂, hydrogen,acetyl, isobutyryl, pivaloyl, and benzoyl, wherein each of R₁, R₂, andR₃ is independently selected from hydrogen and C₁₋₆ alkyl, and wherein nis an integer of from 1 to 10; and directions for administering thepharmaceutical composition to a patient infected with a DNA virus, anRNA virus, or a DNA or RNA reverse transcribing virus.

In an embodiment of the fourth aspect, the kit further comprises anantiviral drug, selected on the basis of its effectiveness for treatingthe given viral agent, in a pharmaceutically acceptable carrier.

In an embodiment of the fourth aspect, the kit further comprises anantiviral drug in a pharmaceutically acceptable carrier and directionsfor administering the antiviral drug in a pharmaceutically acceptablecarrier to the patient.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following description and examples illustrate some exemplaryembodiments of the disclosed invention in detail. Those of skill in theart will recognize that there are numerous variations and modificationsof this invention that are encompassed by its scope. Accordingly, thedescription of a certain exemplary embodiment should not be deemed tolimit the scope of the present invention.

Amifostine

Amifostine is an organic thiophosphate which selectively protects normaltissues but not tumors against cytotoxicity of ionizing radiations,DNA-binding chemotherapeutic agents (e.g., classical alkylating agentssuch as cyclophosphamide and non-classical alkylating agents such asmitomycin-C and platinum analogs). Amifostine is a prodrug that isdephosphorylated to the active metabolite, the free thiol form, byalkaline phosphatase and exits the bloodstream rapidly.

It has surprisingly been discovered that the compounds of preferredembodiments, including amifostine and its derivatives and analogs, areparticularly effective antiviral compounds for use in inhibitingreplication of diverse species of DNA viruses, RNA viruses and DNA andRNA reverse transcribing viruses.

Amifostine (referred to as “WR-2721”) is marketed under the name Ethyol®by Schering-Plough Pty Ltd. It has the chemical nameS-2-(3-aminopropyl)aminoethyl phosphorothioic acid and the structure:

A particularly preferred antiviral compound for use in inhibitingreplication of DNA viruses, RNA viruses and DNA and RNA reversetranscribing viruses includes the free thiol form of amifostine. Thefree thiol form (referred to as “WR-1065”) has the chemical name2-(3-aminopropylamino)ethanethiol and the following structure:

The disulfide form of amifostine (referred to as WR-33278) has thechemical name N¹,N³,(dithiodiethane-2,1-diyl)dipropane-1,3-diamine

Phosphonol (referred to as “WR-3689”) is structurally similar toamifostine, the only difference being the presence of a terminal methylgroup. Phosphonol has the chemical nameS-2-(3-(methylamino)propylamino)ethyl phosphorothioic acid and thefollowing structure:

The free thiol form of phosphonol (referred to as WR-255591) has thechemical name 2-(3-(methylamino)propylamino) ethanethiol and thefollowing structure:

The disulfide form of phosphonol (referred to as WR-33278) has thefollowing structure:

The compounds of preferred embodiments include prodrug forms of theabove-described free thiol forms of amifostine, phosphonol, and analogsthereof. Such prodrugs include compounds of the structure:

wherein each of R₁, R₂, and R₃ is independently selected from hydrogenand lower alkyl, wherein —(C_(n)H_(2n))— is lower alkyl and n is 1, 2,3, 4, 5, or (up to 10), and wherein X is a suitable leaving group.Suitable leaving groups include —PO₃H₂, hydrogen, acetyl, isobutyryl,pivaloyl, and benzoyl); however, any other suitable leaving group can beemployed that yields the active free thiol form of the compound whenmetabolized in vivo. Other leaving groups can include alkyl groups,e.g., —(C₁₋₆ alkyl), and keto groups, e.g., —C(═O)—(C₁₋₆ alkyl) or—C(═O)—(C₆₋₁₈ aryl).

The term “lower alkyl” as used herein is a broad term, and is to begiven its ordinary and customary meaning to a person of ordinary skillin the art (and is not to be limited to a special or customizedmeaning), and refers without limitation to a straight chain or branchedchain, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1,2, 3, 4, 5, or 6 carbon atoms. Saturated straight chain lower alkylsinclude methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl; whilerepresentative saturated branched chain alkyls include isopropyl,sec-butyl, isobutyl, tert-butyl, and isopentyl. “aryl,” as used hereinis a broad term and is used in its ordinary sense, including, withoutlimitation, to refer to an aromatic carbocyclic moiety such as phenyl ornaphthyl, including mono-, di-, and poly-homocyclic aromatic ringsystems (e.g., C₆₋₁₈ aryl).

The prodrug forms described above are metabolized into active thiols ofthe formula:

wherein each of R₁, R₂, R₃, —(C_(n)H₂O—, and n is as defined above. In aparticularly preferred embodiment, —(C_(n)H_(2n))— is a straight alkylchain having three carbon atoms. It is also particularly preferred thatR₂ and R₃ are both hydrogen, and that R₁ is hydrogen or methyl.

In addition to the thiols, certain disulfide forms also exhibitantiviral activity, for example, a disulfide of the following structure:

can also be employed as an antiviral agent.

Other antiviral agents of preferred embodiments incorporate acysteamine-like group (i.e., a group containing the moiety>N—(CH₂)₂—S—), or a moiety with the structure of WR 2721 or WR-1065,tethered to a DNA or nucleic acid binding agent, or other agents withsome affinity for nucleic acids or proteins.

The compounds of preferred embodiments are particularly effectiveantiviral agents for monotherapeutic or combined-therapeutic use intreating DNA viruses, RNA viruses and DNA and RNA reverse transcribingviruses. The compounds of preferred embodiments are generallyadministered at dosages equal to or less than the oral radioprotectivedosage of amifostine (e.g., 1456 mg total dose, or 910 mg/m² for a 60 kgbody weight (BW) adult human) or phosphonol (e.g., 725 mg/m²). In adultsundergoing chemotherapy, the recommended starting dose of amifostine is910 mg/m² for a 60 kg BW adult human administered once daily as a15-minute i.v. infusion, starting 30 minutes prior to chemotherapy.Similar dosing regimens can be employed for use of the compounds ofpreferred embodiments when used as antiviral agents. Dosages can beconverted from mg/m² to total mg or mg/kg BW (see, e.g., Freireich etal. (1966), Cancer Chemother. Reports, 50 (4) 219-244) as in Table 2.

TABLE 2 Species Body Wt. (Kg) Body Surface area (m²) Km factor Mouse 0.20.0066 3.0 Rat 0.15 0.025 5.9 Monkey 3.0 0.24 12 Dog 8.0 0.40 20 Humanchild 20.0 0.80 25 Human adult 60.0 1.60 37

It is generally preferred to administer amifostine (or other compoundsof preferred embodiments) at dosages of 910 mg/m² or less. This dosageis equivalent to 24.3 mg/kg BW for a 60 kg BW adult human being (or atotal dose of 1456 mg for a 60 kg BW adult, as described above);however, in certain embodiments it can be desirable to administer thecompounds of preferred embodiments at higher dosage levels. For example,children have been given up to a 2700 mg/m² total dose of amifostineprior to administration of a chemotherapeutic agent. In some individualssuch high doses are associated with side effects. A dose of 740 mg/m²amifostine (1148 mg total, for a 60 kg BW human adult) is associatedwith fewer side effects (List et al. (1997), Blood 90(9): 3364-9), andis thus generally preferred. For daily dosing, 200-340 mg/m² ofamifostine (544 mg total dose for a 60 kg BW adult) is generallypreferred (Schuchter (1996), Semin Oncol 23(4 Suppl 8): 40-3; Santini etal. (1999), Haematologica 84(11): 1035-42).

Rodent studies suggest the use of higher dosages. For example, themaximally tolerated dose (MTD) for WR-2721 in mice was 432 mg/kg (BW)administered i.p. and 720 mg/kg BW administered p.o., and the 100%effective radioprotective dose was about one half of the MTD. Forphosphonol, the MTD was 893 mg/kg BW administered i.p. and 1488 mg/kg BWadministered p.o., and the 100% effective radioprotective dose was aboutone half of the MTD. All of the aminothiols have MTDs in rodents ofgreater than 400 mg/kg BW.

Amifostine and WR-1065 can be efficacious at very low concentrations,for example, down to 0.4 micromolar concentrations in some in vitrostudies. In a preferred embodiment, a drug delivery system that obtainsrelatively constant intracellular concentrations over a period of timethat could extend from several days to up to one year or more isdesired. To achieve this goal, we anticipate using drug delivery systemsdemonstrated to achieve relatively constant intracellular drug levelsover extended time periods. Examples of such drug delivery systemsinclude subcutaneous administration, formulation in nanoparticles, wereformulation in other slow release systems.

Table 3 provides plasma concentrations of amifostine metabolites (seeGeary et al., Res. Comm. Chem. Path. Pharmacol., 65(2), 147-159 (1989))and phosphonol metabolites (see Buckpitt et al., Contract #DAMD17-86-C-6177, reference obtained from Dr. D. Grdina, personalcommunication) after duodenal administration of 150 mg/kg BW of eachdrug in rhesus macaques. This data was collected as part of workperformed to evaluate the radioprotective activity of the compounds.These data may be useful in estimating plasma concentrations in humans.

TABLE 3 Drug Time after Administration (h) Concentration (μg/ml)WR-1065¹ 1 6.21 WR-1065¹ 2 4.14 WR-1065¹ 6 2.07 Phosphonol² 1 11.75Phosphonol² 2 17.08 Phosphonol² 6 15.50 Note: 40 μM WR-1065 = 8.28 μg/ml

While it is generally preferred to formulate amifostine or the othercompounds of preferred embodiments for oral administration, thecompounds of preferred embodiments can be formulated so as to allow themto be administered by other routes, as discussed herein. It can bedesirable in certain embodiments to formulate amifostine for intravenousadministration in order to maximize efficacy. Because of the structuralsimilarities between amifostine and phosphonol, especially thesimilarities in the sulfhydryl ends of the molecules, phosphonol isexpected to behave in a manner similar to amifostine rather thanWR-151327 (chemical formula CH₃NH(CH₂)₃NH(CH₂)₃SPO₃H₂). WR-151327 hasbeen previously shown to have anti-HIV activity; this activity wasattributed to the ability of the compound to modulate cytokine levels ina manner that inhibited HIV replication and/or proliferation (Kalebic etal., 1994).

Phosphonol may not be quite as efficacious as amifostine, based upon thework of Gutschow et al., who found lower activity when a methyl groupwas substituted for a hydrogen atom in a position similar to that of themethyl group of phosphonol that distinguishes its structure from that ofamifostine (Gutschow et al. (1995), Pharmazie 50(10): 672-5). However,phosphonol is expected to be significantly more efficacious thanWR-151327. This consideration should be viewed in light of the fact thatthe overall structures of the compounds tested by Gutschow et al. weresignificantly different from the structures of the preferredembodiments. The overall structures of the compounds tested by Gutschowet al. are significantly different from the structures of the preferredembodiments, and it is expected that phosphonol will be significantlymore efficacious than WR-151327.

Amifostine, its analogs, and its derivatives can be administered incombination with other antiviral agents employed to treat HIV infection.One of the benefits of such combination therapies is that lower doses ofthe other antiviral agents can be administered while still achieving asimilar level of antiviral efficacy. Such lower dosages can beparticularly advantageous for drugs known to have genotoxicity andmitochondrial toxicity (for example, some nucleoside analogs).Conversely, greater efficacy might be achieved using therapeutic dosesof two drugs than could be achieved using only a single drug.

The most common antiviral drugs that can be used in junction with thecompounds of preferred embodiments include, but are not limited, tomembers of the class of drugs known as nucleoside analogs. Other agentsthat could be used potentially are included in the list of antiviraldrugs included in Appendix 2. The use of nucleoside analogs has beenassociated with a variety of side effects due to the fact that thesedrugs are analogs of naturally occurring nucleosides, are incorporatedinto host cell DNA, and function as obligate DNA chain terminators. Assuch, these drugs are associated with the induction of mutations in hostcell DNA, increased risks for certain types of cancer, as well asincreased risks for a variety of diseases associated with mutationinduction in nuclear and mitochondrial DNA. Nucleoside analogs are alsoassociated with mitochondrial toxicity to a degree that varies dependingupon the specific nucleoside analog in question. Thus, it is desirableto be able to minimize the doses of nucleoside analogs used, and/or touse these drugs in combination with other drugs with demonstratedantiviral efficacy that does not compromise the effectiveness of theother treatment, and that also has demonstrated antimutagenic efficacy.

The use of the compounds of preferred embodiments have been tested incell culture with one nucleoside analog (zidovudine), and it is beendemonstrated that the efficacy of each compound is not diminished whenused in combination with the other. It is anticipated that the compoundsof preferred embodiments can also be effectively employed with othernucleoside analogs and with other antiviral agents (see Appendix 2).

Pharmaceutical Compositions Comprising Amifostine and Analogs Thereof:

The compounds of preferred embodiments (including derivatives, isomers,metabolites, prodrugs, or pharmaceutically acceptable esters, salts, andsolvates thereof) can be incorporated into a pharmaceutically acceptablecarrier, including incorporation into nanoparticles for administrationto an individual having a viral infection (for example, an adenoviralinfection as an example of a DNA virus, an influenza infection as anexample of a RNA virus, or an HIV infection as an example of a DNA orRNA reverse transcribing virus) or can be administered prophylacticallyto prevent viral infection, or postinfection to decrease symptoms uponviral infection. The compounds of preferred embodiments can be employedas the sole agent in the prevention or treatment of DNA viruses, RNAviruses, or DNA or RNA reverse transcribing viruses, and/or combinationsthereof two or more such compounds can be employed, optionally incombination with other therapeutic agents, e.g., conventional or newlydeveloped drugs employed in the treatment of AIDS or HIV, or other viralinfections.

Because certain of the compounds of preferred embodiments can besensitive to oxidation, it can be desirable to administer the compoundsin combination with reducing agents including, but not limited to,vitamin C and vitamin E. Other reducing agents include organicaldehydes, hydroxyl-containing aldehydes, and reducing sugars such asglucose, mannose, galactose, xylose, ribose, and arabinose. Otherreducing sugars containing hemiacetal or keto groupings can be employed,for example, maltose, sucrose, lactose, fructose, and sorbose. Otherreducing agents include alcohols, preferably polyhydric alcohols, suchas glycerol, sorbitol, glycols, especially ethylene glycol and propyleneglycol, and polyglycols such as polyethylene and polypropylene glycols.

The terms “pharmaceutically acceptable salts” and “a pharmaceuticallyacceptable salt thereof” as used herein are broad terms, and are to begiven their ordinary and customary meaning to a person of ordinary skillin the art (and are not to be limited to a special or customizedmeaning), and refer without limitation to salts prepared frompharmaceutically acceptable, non-toxic acids or bases. Suitablepharmaceutically acceptable salts include metallic salts, e.g., salts ofaluminum, zinc, alkali metal salts such as lithium, sodium, andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts; organic salts, e.g., salts of lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), procaine, and tris;salts of free acids and bases; inorganic salts, e.g., sulfate,hydrochloride, and hydrobromide; and other salts which are currently inwidespread pharmaceutical use and are listed in sources well known tothose of skill in the art, such as, for example, The Merck Index. Anysuitable constituent can be selected to make a salt of the therapeuticagents discussed herein, provided that it is non-toxic and does notsubstantially interfere with the desired activity. In addition to salts,pharmaceutically acceptable precursors and derivatives of the compoundscan be employed. Pharmaceutically acceptable amides, lower alkyl esters,and protected derivatives can also be suitable for use in compositionsand methods of preferred embodiments. While it may be possible toadminister the compounds of the preferred embodiments in the form ofpharmaceutically acceptable salts, it is generally preferred toadminister the compounds in neutral form.

It is generally preferred to administer the compounds of preferredembodiments orally; however, other routes of administration arecontemplated. Contemplated routes of administration include but are notlimited to oral, sublingual, parenteral, transcutaneous, subcutaneous,intravenous, and by inhalation. Compounds of preferred embodiments canbe formulated into liquid preparations for, e.g., oral administration.Suitable forms for such administration include suspensions, syrups, andelixirs.

The pharmaceutical compositions of preferred embodiments are preferablyisotonic with the blood or other body fluid of the recipient. Theisotonicity of the compositions can be attained using sodium tartrate,propylene glycol or other inorganic or organic solutes. Sodium chlorideis particularly preferred. Buffering agents can be employed, such asacetic acid and salts, citric acid and salts, boric acid and salts, andphosphoric acid and salts. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. In certain embodiments it can be desirable tomaintain the active compound in the reduced state. Accordingly, it canbe desirable to include a reducing agent, such as vitamin C, vitamin E,or other reducing agents as are known in the pharmaceutical arts, in theformulation.

Viscosity of the pharmaceutical compositions can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the thickening agentselected. An amount is preferably used that will achieve the selectedviscosity. Viscous compositions are normally prepared from solutions bythe addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf life of the pharmaceutical compositions. Benzyl alcohol can besuitable, although a variety of preservatives including, for example,parabens, thimerosal, chlorobutanol, or benzalkonium chloride can alsobe employed. A suitable concentration of the preservative is typicallyfrom about 0.02% to about 2% based on the total weight of thecomposition, although larger or smaller amounts can be desirabledepending upon the agent selected. Reducing agents, as described above,can be advantageously used to maintain good shelf life of theformulation.

The compounds of preferred embodiments can be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, or the like, and can contain auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,gelling or viscosity enhancing additives, preservatives, flavoringagents, colors, and the like, depending upon the route of administrationand the preparation desired. See Standard texts, such as “Remington: TheScience and Practice of Pharmacy”, Lippincott Williams & Wilkins; 20thedition (Jun. 1, 2003) and “Remington's Pharmaceutical Sciences,” MackPub. Co.; 18^(th) and 19^(th) editions (December 1985, and June 1990,respectively). Such preparations can include complexing agents, metalions, polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, and the like, liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. The presence of suchadditional components can influence the physical state, solubility,stability, rate of in vivo release, and rate of in vivo clearance, andare thus chosen according to the intended application, such that thecharacteristics of the carrier are tailored to the selected route ofadministration.

For oral administration, the pharmaceutical compositions can be providedas a tablet, aqueous or oil suspension, dispersible powder or granule,emulsion, hard or soft capsule, syrup or elixir. Compositions intendedfor oral use can be prepared according to any method known in the artfor the manufacture of pharmaceutical compositions and can include oneor more of the following agents: sweeteners, flavoring agents, coloringagents and preservatives. Aqueous suspensions can contain the activeingredient in admixture with excipients suitable for the manufacture ofaqueous suspensions.

Formulations for oral use can also be provided as hard gelatin capsules,wherein the active ingredient(s) are mixed with an inert solid diluent,such as calcium carbonate, calcium phosphate, or kaolin, or as softgelatin capsules. In soft capsules, the active compounds can bedissolved or suspended in suitable liquids, such as water or an oilmedium, such as peanut oil, olive oil, fatty oils, liquid paraffin, orliquid polyethylene glycols. Stabilizers and microspheres formulated fororal administration can also be used. Capsules can include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredient in admixture with fillerssuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In instanceswhere it is desirable to maintain a compound of a preferred embodimentin a reduced form (in the case of certain active metabolites), it can bedesirable to include a reducing agent in the capsule.

Tablets can be uncoated or coated by known methods to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period of time. For example, atime delay material such as glyceryl monostearate can be used. Whenadministered in solid form, such as tablet form, the solid formtypically comprises from about 0.001 wt. % or less to about 50 wt. % ormore of active ingredient(s), preferably from about 0.005, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, or 45 wt. %.

Tablets can contain the active ingredients in admixture with non-toxicpharmaceutically acceptable excipients including inert materials. Forexample, a tablet can be prepared by compression or molding, optionally,with one or more additional ingredients. Compressed tablets can beprepared by compressing in a suitable machine the active ingredients ina free-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding, in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

Preferably, each tablet or capsule contains from about 10 mg or less toabout 1,000 mg or more of a compound of the preferred embodiments, morepreferably from about 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg to about150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or900 mg. Most preferably, tablets or capsules are provided in a range ofdosages to permit divided dosages to be administered. A dosageappropriate to the patient and the number of doses to be administereddaily can thus be conveniently selected. In certain embodiments it canbe preferred to incorporate two or more of the therapeutic agents to beadministered into a single tablet or other dosage form (e.g., in acombination therapy); however, in other embodiments it can be preferredto provide the therapeutic agents in separate dosage forms.

Suitable inert materials include diluents, such as carbohydrates,mannitol, lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans, starch, and the like, or inorganic salts such as calciumtriphosphate, calcium phosphate, sodium phosphate, calcium carbonate,sodium carbonate, magnesium carbonate, and sodium chloride.Disintegrants or granulating agents can be included in the formulation,for example, starches such as corn starch, alginic acid, sodium starchglycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose,natural sponge and bentonite, insoluble cationic exchange resins,powdered gums such as agar, karaya or tragacanth, or alginic acid orsalts thereof.

Binders can be used to form a hard tablet. Binders include materialsfrom natural products such as acacia, tragacanth, starch and gelatin,methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, and the like.

Lubricants, such as stearic acid or magnesium or calcium salts thereof,polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes,sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol,starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like,can be included in tablet formulations.

Surfactants can also be employed, for example, anionic detergents suchas sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctylsodium sulfonate, cationic such as benzalkonium chloride or benzethoniumchloride, or nonionic detergents such as polyoxyethylene hydrogenatedcastor oil, glycerol monostearate, polysorbates, sucrose fatty acidester, methyl cellulose, or carboxymethyl cellulose.

Controlled release formulations can be employed wherein the amifostineor analog(s) thereof is incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms. Slowly degeneratingmatrices can also be incorporated into the formulation. Other deliverysystems can include timed release, delayed release, or sustained releasedelivery systems.

Coatings can be used, for example, nonenteric materials such as methylcellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethylcellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose,sodium carboxy-methyl cellulose, providone and the polyethylene glycols,or enteric materials such as phthalic acid esters. Dyestuffs or pigmentscan be added for identification or to characterize differentcombinations of active compound doses.

When administered orally in liquid form, a liquid carrier such as water,petroleum, oils of animal or plant origin such as peanut oil, mineraloil, soybean oil, or sesame oil, or synthetic oils can be added to theactive ingredient(s). Physiological saline solution, dextrose, or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol, or polyethylene glycol are also suitable liquid carriers. Thepharmaceutical compositions can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil, such as olive orarachis oil, a mineral oil such as liquid paraffin, or a mixturethereof. Suitable emulsifying agents include naturally-occurring gumssuch as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsions can also containsweetening and flavoring agents.

When a compound of the preferred embodiments is administered byintravenous, parenteral, or other injection, it is preferably in theform of a pyrogen-free, parenterally acceptable aqueous solution oroleaginous suspension. Suspensions can be formulated according tomethods well known in the art using suitable dispersing or wettingagents and suspending agents. The preparation of acceptable aqueoussolutions with suitable pH, isotonicity, stability, and the like, iswithin the skill in the art. A preferred pharmaceutical composition forinjection preferably contains an isotonic vehicle such as1,3-butanediol, water, isotonic sodium chloride solution, Ringer'ssolution, dextrose solution, dextrose and sodium chloride solution,lactated Ringer's solution, or other vehicles as are known in the art.In addition, sterile fixed oils can be employed conventionally as asolvent or suspending medium. For this purpose, any bland fixed oil canbe employed including synthetic mono or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the formation ofinjectable preparations. The pharmaceutical compositions can alsocontain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

The duration of the injection can be adjusted depending upon variousfactors, and can comprise a single injection administered over thecourse of a few seconds or less, to 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours or more of continuous intravenous administration.

The antiviral compositions of the preferred embodiments can additionallyemploy adjunct components conventionally found in pharmaceuticalcompositions in their art-established fashion and at theirart-established levels. Thus, for example, the compositions can containadditional compatible pharmaceutically active materials for combinationtherapy (such as supplementary antimicrobials, antipruritics,astringents, local anesthetics, anti-inflammatory agents, reducingagents, and the like), or can contain materials useful in physicallyformulating various dosage forms of the preferred embodiments, such asexcipients, dyes, thickening agents, stabilizers, preservatives orantioxidants.

The compounds of the preferred embodiments can be provided to anadministering physician or other health care professional in the form ofa kit. The kit is a package which houses a container which contains thecompound(s) in a suitable pharmaceutical composition, and instructionsfor administering the pharmaceutical composition to a subject. The kitcan optionally also contain one or more additional therapeutic agents,e.g., antiviral nucleoside analog, peptide analog, protease inhibitor,monoclonal antibody, or any other antiviral used for the treatment orprevention of virus disease (see for example Appendix 2). For example, akit containing one or more compositions comprising compound(s) of thepreferred embodiments in combination with one or more additionalantiviral, antibacterial, and/or anti-infective agents can be provided,or separate pharmaceutical compositions containing a compound of thepreferred embodiments and additional therapeutic agents can be provided.The kit can also contain separate doses of a compound of the preferredembodiments for serial or sequential administration. The kit canoptionally contain one or more diagnostic tools and instructions foruse. The kit can contain suitable delivery devices, e.g., syringes, andthe like, along with instructions for administering the compound(s) andany other therapeutic agent. The kit can optionally contain instructionsfor storage, reconstitution (if applicable), and administration of anyor all therapeutic agents included. The kits can include a plurality ofcontainers reflecting the number of administrations to be given to asubject. In a particularly preferred embodiment, a kit for the treatmentof DNA viruses, RNA viruses, and DNA and RNA reverse transcribingviruses, and combinations thereof is provided that includes amifostineor another compound of a preferred embodiment and one or more antiviralagents currently employed to treat the virus. Antiviral agents includefor example, nucleoside analogs such as acyclovir, reverse transcriptaseinhibitors such as zidovudine, didanosine, zalcitabine, stavudine, 3TC;non-nucleoside reverse transcriptase inhibitors; protease inhibitors;cytokines; immunomodulators, and antibodies but are not limited thereto.

The compounds of the preferred embodiments can be administeredprophylactically for the prevention of DNA virus, RNA virus, or DNA orRNA reverse transcribing virus infection. Alternatively, therapy ispreferably initiated as early as possible following the onset of signsand symptoms of a viral infection, or following exposure to a DNA virus,a RNA virus, or a DNA or RNA reverse transcribing virus infection. Theadministration route, amount administered, and frequency ofadministration will vary depending on the age of the patient, theseverity of the infection, and any associated conditions. Contemplatedamounts, dosages, and routes of administration for the compounds ofpreferred embodiments for treatment of a DNA virus, a RNA virus, or aDNA or RNA reverse transcribing virus infection are similar to thoseestablished for conventional antiviral agents. Detailed informationrelating to administration and dosages of conventional antiviral agentscan be found in the Physician's Desk Reference, 47th edition. Thisinformation can be adapted in designing treatment regimes utilizing thecompounds of preferred embodiments. It is expected that anti-viraltherapies combining an aminothiol with another antiviral therapy willhave an additive or synergistic activity, making it possible to reducethe doses of the drugs currently used to treat DNA virus, RNA virus, orDNA or RNA reverse transcribing virus infection, or making it possibleto achieve higher efficacy by using therapeutic doses of two or moreagents together. The combination of an aminothiol with a nucleosideanalog is expected to simultaneously provide improved antiviral therapyand protection against nucleoside analog-induced side effects,especially those associated with nuclear DNA or mitochondrial DNAdamage. It is further anticipated that an aminothiol could protectagainst deleterious side effects of another antiviral drugs, especiallythose that induce nuclear DNA or mitochondrial DNA damage.

According to one embodiment, contemplated amounts of the compounds ofthe preferred embodiments for oral administration to treat DNA virus,RNA virus, or DNA or RNA reverse transcribing virus infection are fromabout 10 mg or less to about 2000 mg or more administered from aboutevery 4 hours or less to about every 6 hours or more (or from about 4times daily to about 6 times daily) for about 5 days or less to about 10days or more (40 mg/day or less to about 15,000 mg/day or more) or untilthere is a significant improvement in the condition. For suppressivetherapy to inhibit the onset of infection in susceptible individuals,doses of from about 10 mg or less to about 1000 mg or more are orallyadministered once, twice, or multiple times a day, typically for up toabout 12 months, or, in certain circumstances, indefinitely (from about10 mg/day to about 1,000 mg/day). When treatment is long term, it can bedesirable to vary the dosage, employing a higher dosage early in thetreatment, and a lower dosage later in the treatment.

The single highest dose of amifostine administered to an adult human asdocumented in the literature was 1330 mg/m². Children have beenadministered single doses of amifostine of up to 2700 mg/m² with nountoward effects. The literature indicates that multiple doses (up tothree times the recommended single dose of 740 to 910 mg/m²) have beensafely administered within a 24-hour period. Repeated administration ofamifostine at two and four hours after the initial dose does not appearto result in an increase in side effects, especially nausea, vomiting,or hypotension. It appears that the most significant deleterious sideeffect from the administration of amifostine is hypotension.

According to one embodiment, contemplated amounts of the compounds ofthe preferred embodiments, methods of administration, and treatmentschedules for individuals with infections caused by DNA viruses, RNAviruses and DNA and RNA reverse transcribing viruses are generallysimilar to those described above for the prevention of and/or treatmentof radiation- or chemotherapy-induced cytotoxicity; they may also besimilar to those used for the treatment of myelodysplastic syndrome.

Known side effects of amifostine include decrease in systolic bloodpressure, nausea, and vomiting. If such side effects are observed forthe particular thiophosphate administered, it is generally preferred toadminister an antiemetic medication prior to, or in conjunction with thethiophosphate. Suitable antiemetic medications include antihistamines(e.g., buclizine, cyclizine, dimenhydrinate, diphenhydramine,meclizine), anticholinergic agents (e.g., scopolamine), dopamineantagonists (e.g., chlorpromazine, droperidol, metoclopramide,prochlorperazine, promethazine), serotonin antagonists (e.g.,dolasetron, granisetron, ondansetron), or other agents (e.g.,dexamethasone, methylprednisolone, trimethobenzamide).

The purity of the WR-1065 used in the experiments described below isunknown. WR-1065 is sensitive to oxidation (and possibly otherreactions) and can undergo reaction to forms that appear to lackantiviral activity. Accordingly, in the tables below and in thepreferred dosages provided above, the indicated concentrations representthe maximum amount of active compound that could be present; the trueconcentration of active compound is less than that indicated but cannotbe determined definitively. In addition, for concentrations of WR-1065of less than 100 μM, estimates of the true efficacy of the compound arelimited by the fact that the compound has been found to be inactivatedby a variety of medium components (Grdina et al. (2000), Drug MetabolDrug Interact 16(4): 237-79). Below approximately 50 μM, this problembecomes especially severe.

The impact of WR1065 treatment on replication on lung prototypicadenovirus strains in A549 cell monolayers is illustrated in Table 4.A549 or MDCK cells were used for the plaque reduction assays. Cells weregrown to near confluence in Minimal Essential Medium (MEM) in six-wellplates. Cells were infected with 100 plaque forming units (PFUs) ofvirus of virus in 100 μl and adsorption was allowed for one hour at 37°C. Cell monolayers were subsequently overlaid with 0.6% agarose in MEM.The overlay medium was prepared to contain a final concentration of 10μM, 33 μM and 100 μM amount of WR 1065. Each concentration was assayedin triplicate. Wells overlaid with media without WR-1065 were set up as100% infectious virus yield controls. Cultures were maintained at 37° C.in 5% CO₂ for 7 days. At the end of this time period, cell cultures werefixed with 1% formaldehyde and stained with Crystal Violet forvisualization of plaques and evaluation of plaque size and number.Reduction of plaque formation was expressed as a percentage of theuntreated controls. Subsequently, WR 1065 was demonstrated to inhibitviral replication and development of cytotopathic effect in adose-dependent manner in influenza A or influenza B-infected MDCK cellmonolayers. Using an approach similar to that described above, MDCKcells growing in 6-well plates were infected with 100 plaque formingunits (PFUs) of virus in 100 μl and adsorption was allowed for one hourat 37° C. Cell monolayers were subsequently overlaid with 0.6% agarosein MEM. The overlay medium was prepared to contain a final concentrationof 10 μM, 33 μM and 100 μM amount of WR 1065. Each concentration wasassayed in triplicate. Wells overlaid with media without WR-1065 wereset up as 100% infectious virus yield controls. Cultures were maintainedat 37° C. in 5% CO₂ for 3 days. At the end of this time period, cellcultures were fixed with 1% formaldehyde and stained with Crystal Violetfor visualization of plaques and evaluation of plaque size and number.Reduction of plaque formation was expressed as a percentage of theuntreated controls.

TABLE 4 Mean infectious virus yields (PFUs × 10⁶) 96-h post infection &WR 1065 treatment^(a) Susceptible 0 μM <10 μM <33 μM <100 μM VirusSpecies populations Trial # WR 1065 WR 1065 WR 1065 WR 1065 Ad7-p BPediatric & military; 1 3.5 1.85 0.85 1.15 (strain respiratory infection2 58.2 — — 30.5 Gomen) Ad5-p C Immuno-compromised; 1 12.5 9.0  5.65 2.25(strain upper respiratory 2 665 — 600 500 Adenoid 3 1,920 — 1,660 — 75)Ad4-p E Military; conjunctivitis & 1 1,900 — 1,420 980 (strain RI-respiratory infection 2 22.5 — — 9.5 67)

Table 5 includes data from an experiment to measure WR-1065 protectionagainst cytopathic effects in MDCK cell monolayers infected withinfluenza A/Puerto Rico/8/34 (H1N1), using densitometry readings of thestained cell monolayers to estimate relative cell survival. Similarresults were obtained in a second independent experiment using influenzaA/Puerto Rico/8/34 (H1N1), and in an experiment using the same approachwith influenza B/Lee/40. In a third experiment with influenza A, WR 1065was added to an agarose overlay following a one-hour adsorption with adifferent virus strain, A/HKx31 (H3N2), and a plaque reduction assay wasperformed (Table 5). The results illustrate protection of MDCK cellsfrom Influenza A by WR 1065, based upon densitometry readings(experiment 1) or a plaque reduction assay (experiment 2)

According to one aspect of the present invention, the reduced form ofamifostine (WR 2721 or ethyol), was the most active moiety in theobserved antiviral effects. In another aspect of the present invention,WR-1065, was the most active moiety in the observed antiviral effects. Acommon medium component (phenylalanine) blocks the activity of WR 1065to some undetermined degree.

TABLE 5 Influenza A strain; Amount of WR 1065^(a) Quantitative approach0 μM <10 μM <33 μM <100 μM Experiment 1 A/Puerto 9.57 ± 12.4 ± 13.1 ±1.0 × 14.3 ± Rico/8/34(H1N1); 0.3 × 10⁵ 0.4 × 10⁵ 10⁵ 1.5 × 10⁵Densitometry reading^(b) Experiment 2 A/HKx31(H3N2); 66.3 59.3 57 48Mean plaque number Densitometry readings = mean counts/mm² ± standarddeviation using Quantity One (Bio-Rad) plate reader.

Protection of A549 cells from the cytopathic effects of Adenovirus 5p byWR-1065 as assessed by a plaque reduction assay is illustrated in Table6.

TABLE 6 Amount of WR1065 0 μM 16.5 μM 50 μM Adenovirus 5p 64 63 41 (36%reduction)

Experiments were conducted to determine the antiretroviral effects ofAZT in the presence of WR-1065, the active metabolite of amifostine.Peripheral blood mononuclear cells (PBMCs) obtained from the NIH BloodBank were cultured with phytohemagglutinin (PHA) for 48 hours to createPHA blasts. The cells were cultured in RPMI-1640 medium with 10% fetalbovine serum (FBS), 1% penicillin/streptomycin and glutamine, and 10%Interleukin-2 (IL-2). The PHA blasts were then infected with HIV for twohours (see Perno et al. (1988), J. Exptl. Med. 168:111; Aquaro et al.(1988), J. Medical Virology 68:479-488).

Noting the caveats outlined above regarding purity of the WR-1065 used,WR-1065 (from the NCI Chemical Carcinogen Repository; see Hoffman et al.(2001), Env. Mol. Mut. 37:117) was weighed (Mol. Wt. 134) and storedfrozen in RPMI-1640 medium as a 10 mg/ml solution (13.4 μL of thesolution added to 1 ml of RPMI-1640 yields 1000 μM solution). AZT(SIGMA, Mol. Wt. 267) was dissolved in phosphate-buffered saline (PBS)at a concentration of 36 mM (9.72 mg/ml), 0.01 ml (97 μg) of theresulting solution was added to 3.6 ml RPMI-1640 medium to yield a 26.9μg/ml solution, and 0.1 ml (2.69 μg) of that resulting solution wasadded to 1 ml of RPMI-1640 medium to yield a 10 μM solution of AZT. TheHIV-infected PHA blasts were incubated with 10 μM AZT in the presence orabsence of 1000 μM WR-1065 prepared as described above.

At 72 hours, HIV infection status was monitored in five experimentalgroups by measuring p24 using an ELISA kit (RETRO-TEK HIV-1, p24Extended Range ELISA, ZMC catalog #0801137). The HIV infection statusfor the five experimental groups is provided in Table 3 (see Experiment#1 data).

TABLE 7 Estimated 10 μM HIV P24 percent viral Treatment Group AZT WR1065pg/ml inhibition Experiment #1 PHA blasts No No 0.28 N.A. PHA blasts +HIV No No 498.62 N.A. PHA blasts + HIV Yes No 0.13 ~100%  PHA blasts +HIV Yes Yes (1000 μM) 0.13 ~100%  PHA blasts + HIV No Yes (1000 μM) 0.13~100%  Experiment #2 PHA blasts No No 8.99 N.A. PHA blasts + HIV No No176.45 N.A. PHA blasts + HIV Yes No 9.07 94.9% PHA blasts + HIV No Yes(1000 μM) 8.92 94.9% PHA blasts + HIV No Yes (330 μM) 9.31 94.7% PHAblasts + HIV No Yes (100 μM) 8.92 94.9% Experiment #3 PHA blasts No No3.0 N.A. PHA blasts + HIV No No 44,455.8 N.A. PHA blasts + HIV Yes No2.9 ~100%  PHA blasts + HIV No Yes (66 μM) 33.2 99.9% PHA blasts + HIVNo Yes (33 μM) 503.2 98.9% PHA blasts + HIV No Yes (10 μM) 911.2 98.0%PHA blasts + HIV No Yes (5 μM) 10,293.7 76.8%

Table 7 illustrates the antiviral efficacy of AZT and WR-1065 on PHAblasts. Subsequent experiments confirmed this antiretroviral effect, anddemonstrated that the ability of WR 1065 to inhibit viral replicationwas dose-dependent and comparable in concentration and effect to thatseen with AZT alone using this in vitro assay system.

The results of these experiments illustrate that WR-1065 and somestructurally-related aminothiols function as broad-spectrum antiviralagents. Taken together, these results further support the hypothesisthat the antiviral activity of WR-1065 is due to its ability to bind tospecific sites on viral RNA, because the only known element common tothe tested viruses is their requirement for the formation and use of RNAduring viral replication (Table 7).

Viability of Human Cells in the Presence of WR-1065

Experiments were conducted to determine the viability of cells exposedto various concentrations of WR-1065. Tests were conducted on PHA blastscultured as described for the previous experiments, but were notincubated in the presence of HIV. WR-1065 was prepared from a 10 mg/mlsolution in RPMI-1640 medium and diluted to the desired concentrations.Cell viability was measured 72 hours after exposure to WR-1065.Percentage of viable cells (compared to unexposed controls) is providedin Table 8.)

TABLE 8 WR-1065 Concentration % Viable Cells (μM) (compared to control)1000 30 500 73 100 63 50 89 10 90 5 82 1 83

The test results demonstrated acceptable cell viability for allconcentrations tested, and particularly good cell viability forconcentrations of 50 μM and lower.

Comparison of Anti-Viral Activity of Cysteamine and WR-1065

Cysteamine (chemical formula H₂NCH₂CH₂SH) has been demonstrated to haveanti-HIV activity (Ho et al. (1995), AIDS Res Hum Retroviruses 11(4):451-9; Bergamini et al. (1996), J Infect Dis 174(1): 214-8). Using an invitro assay system as described above, 200 μM cysteamine effectivelysuppressed (˜100%) HIV replication. In comparison, WR-1065 effectivelysuppressed (>99.9%) HIV replication at a concentration of less than 100μM. Thus, the in vitro anti-HIV activity of WR-1065 is over 2-foldhigher than that of cysteamine. In addition, cysteamine's duration ofaction was determined to be very short; to achieve an anti-HIV effect,fresh cysteamine had to be added to the cell culture system every 12hours. WR-1065, however, had a long duration of action—it only had to beadded to the culture system once in a 72 hour period.

Comparison of Anti-Viral Activity of Cystamine and WR-1065

Cystamine (chemical formula H₂N(CH₂)₂SS(CH₂)₂NH₂), the oxidized form ofcysteamine, has been demonstrated to have anti-HIV activity, DNA bindingcapacity, radioprotective capacity, and the ability to shift theequilibrium of DNA from the A-form towards the B-form (Allegra et al.(2002), Amino Acids 22(2): 155-66). WR-1065, the active form of WR-2721,has also been shown to bind to DNA in the minor groove, and also toshift the B/A-DNA equilibrium towards the B-form. Accordingly, it can beinferred that cystamine and its reduced form cysteamine are capable ofbinding to other nucleic acids in addition to DNA, and have some abilityto bind to and/or to interact with proteins. It should be noted that theanti-HIV activity of cystamine is considered to be due, at least inlarge part, to its rapid in vivo conversion to cysteamine.

A comparison of the DNA phosphate binding capacity of cystamine versusWR-1065 demonstrated that these two compounds have similar bindingaffinities under similar in vitro conditions (Smoluk et al. (1986),Radiat Res. 107(2):194-204). However, WR-1065 is a larger molecule thancysteamine because it contains an additional moiety: —(CH₂)₃NH₂. Thus,it can be hypothesized that WR-1065 can bind to and thereby block alarger fragment of a nucleic acid than cysteamine. It can therefore behypothesized that the differential binding characteristic of WR-1065versus cysteamine may be responsible for WR-1065's improved antiviralefficacy. It is possible that WR-1065's antiviral activity is due inpart to its ability to bind to and to block critical sites on nucleicacids and/or proteins. (Allegra et al. (2002), Amino Acids 22(2):155-66; North et al., (2002), Mol. Carcinog. 33(3): 181-8). Blockage ofnucleic acid sites and/or proteins is considered to be a possiblemechanism for the aminothiols' modulation of enzyme function (Brekken etal. (1986), J Biol Chem 273(41): 26317-22). The mechanism by which anaminothiol could bind to and/or block nucleic acids and/or proteinswithout inducing significant cytotoxicity to eukaryotic cells is unknownat this time.

As further evidence of the potential importance of WR-1065's nucleicacid binding capacity to its antiviral activity, it has beendemonstrated that WR-1065 has antiviral efficacy against three differentspecies of adenovirus and two different strains of influenza. These aredramatically different types of viruses with significantly differentmodes of replication. One common replication element shared by all ofthese viruses, as well as HIV, is a requirement for single ordouble-stranded RNA during part of the replication cycle. Thus, thedemonstration of broad-spectrum antiviral activity supports thehypothesis that the ability of the antiviral agent WR-1065 to bind toRNA plays a role in the observed antiviral effect.

DISCUSSION

While not wishing to be bound by any particular theory, it is believedthat one or more of the compounds of the present invention function asan antiviral agent because its structure renders it bi-functional,combining two distinct properties. For example, the —(CH₂)₃NH₂ portionof WR-1065 is believed to be responsible for binding to nucleic acids(DNA, RNA), and possibly also to some proteins. A second portion ofWR-1065 —NH(CH₂)₂SH is believed to be largely responsible for theantiretroviral/antiviral effects that have been observed To be effectiveagainst some viruses, it is believed that the sulfhydryl group needs tobe in the reduced state in order for antiretroviral/antiviral effects tobe observed for the compound; however, it is possible that thesulfhydryl group may remain functional if oxidized to the disulfide (asin WR-33278). Both of these components contribute to maximizingantiretroviral/antiviral effects of the compounds of preferredembodiments (e.g., both the —(CH₂)₃NH₂ and the —NH(CH₂)₂SH moieties ofamifostine). According to one embodiment, the first portion of themolecule functions to align the molecule in close proximity to criticalbinding sites on nucleic acids and/or proteins, and the second portionof the molecule functions in a reaction that contributes to antiviralefficacy.

The above hypothesis for the mode of action of WR-1065 and relatedcompounds is in part based upon several compounds have been founddescribed to inhibit replication of multiple different families ofviruses (Qian-Cutrone et al., 1996, Hamasaki and Ueno, 2001, Lacourciereet al. 2000, Li et al. 2001, Nishizono and Nair, 2000, Zang and Yen,1999, Reddy et al. 1999, Xiao et al., 2001). These compounds werehypothesized to bind to and/or otherwise block post-transcriptionalregulatory elements such as the REV response element (RRE) of HIV-1 orthe PRE binding site of hepatitis B virus. These elements are requiredfor export of viral mRNA from the nucleus to the cytoplasm, formation ofviral proteins, and assembly of viral components. Some compounds arehypothesized to bind directly to the RRE or PRE; others are hypothesizedto interfere with protein-RNA binding through indirect mechanisms. Mostof the studied compounds were known to be structurally related to thebroad category of cellular compounds known as polyamines and/or to havepolyamine-like functions. Polyamines are ubiquitous, naturally occurringcompounds found in all cells that are hypothesized to bind to DNA andRNA and to be involved in gene expression regulation; thus, thehypothesized mode of action for the antiviral effects of these agents isconsistent with their structure and what is known about their naturallyoccurring counterparts. It should be noted that amifostine and itsanalogs differ significantly in structure from the above describedcompounds.

Variations to the chemical structure of the moiety —(CH₂)₃NH₂ may betolerated without losing or altering the antiretroviral/antiviralefficacy of the compound, so long as the resulting structure retains abinding capacity/affinity that is similar to that observed with—(CH₂)₃NH₂ itself. Such variations may include more or less than threecarbon atoms in the alkyl group, and a branched alkyl chain, lower alkylsubstituents on the amino group. The potential importance of the moiety—(CH₂)₃NH₂ (or suitable variant) to antiviral effectiveness of thecompounds of preferred embodiments is supported by the work of Laayounet al., who demonstrated a significant increase in anti-mutagenicactivity when cysteamine or WR-2721 is tethered to a chromophore(quinoline or acridine) that increases DNA binding (Laayoun et al.(1994), Int J Radiat Biol 66(3): 259-66). It is hypothesized that theantimutagenic activity of the aminothiols may result from a mode ofaction that is also relevant for their antiviral activity, suggestingthat viral affinity could be altered by changing the chemical structureof the moiety —(CH₂)₃NH₂.

Gutschow et al. describe two compounds that are demonstrated to displaysignificant antiretroviral/antiviral activity (Gutschow et al. (1995),Pharmazie 50(10): 672-5). These compounds differ in structureconsiderably from the compounds of preferred embodiments, but the twomost efficacious compounds have, as a portion of their structure, acysteamine-like group. Gutschow et al. tested a number of compounds,some of which differed in the number of carbon atoms separating thesulfhydryl group and its adjacent amino group, and noted that theoptimal antiviral effect was observed using molecules that had acysteamine-like group and that this effect was diminished if the numberof —CH₂— groups separating the sulfhydryl group and the first aminogroup was increased to three, as occurs in the aminothiols WR-151327(chemical formula CH₃NH(CH₂)₃NH(CH₂)₃SPO₃H₂) and WR-151326 (chemicalformula CH₃NH(CH₂)₃NH(CH₂)₃SH). Accordingly, altering the structure ofthe cysteamine-like group to include three —CH₂— groups between thesulfhydryl group and the amino group significantly reduced the capacityof the compound to function as an antiretroviral/antiviral agent.

In contrast to WR-151327 and WR-151326, other compounds of preferredembodiments have only two —CH₂— groups separating the sulfhydryl groupfrom the first amino group, and thus possess superiorantiretroviral/antiviral properties. The antiretroviral/activity ofWR-1065 is significantly greater than that reported for WR-151326, andthus WR-1065 has functional capacities that are different from those ofWR-151327 and WR-151326.

Significant questions remain as to how a molecule such as WR-1065 couldinteract with viral nucleic acids and proteins in such a way as toinhibit critical processes (such as replication, transcription,translation, or protein aggregation/functionality) withoutsimultaneously exerting a similar effect upon cells of higher organisms.Some information from early work performed with viral genomes as modelsof DNA replication can be used to formulate testable hypotheses.

We hypothesize that for example, WR-1065 and its analogs interfere withcritical components of the viral life cycle of many/all viruses throughinteractions with viral nucleic acids, nucleic acid-associated proteins,and/or molecular machines involved in nucleic acid and/or proteinproduction and maintenance. We further hypothesize that fundamentaldifferences in nucleic acid and protein production and maintenance ofviruses versus higher organisms are responsible for the differentialeffects of WR-1065 and its analogs. The rationale for these hypothesesis supported by the following information.

Experimental evidence supports the hypothesis that the nucleic acids ofhigher organisms have a protein-to-DNA ratio of approximately 1:10. Incontrast, the nucleic acids of many viruses, vegetative bacteriophage,and dinoflagellates have a protein-to-DNA ratio of approximately 1:1(Cremisi, 1979; Kellenberger, 1988). The significantly higher ratio ofproteins to nucleic acids of viruses would allow for enhancedinteraction between proteins associated with nucleic acids, the nucleicacids themselves, and a molecule with nucleic acid and protein bindingaffinity. The enhanced opportunity for binding between WR-1065 and viralnucleic acids and proteins could result in deleterious effects uponviral nucleic acid production, maintenance, or functionality. A similareffect would not be expected to be exerted at the same magnitude uponthe nucleic acids of higher organisms. The significantly lower ratio ofproteins to nucleic acids in higher organisms versus viruses would beexpected to reduce the opportunities for interaction/binding betweenWR-1065, nucleic acids, and adjacent proteins in higher organisms by adegree roughly comparable to the differences in the ratios of protein tonucleic acid found in these two different life forms.

In addition, there are considerable differences in the types and amountsof proteins associated with viral nucleic acids as opposed to thenucleic acids of higher organisms (Cremisi, 1979). These protein-baseddistinctions could result in further differences in the number or typesof interactions between WR-1065 and components of viruses versuscellular components of higher organisms.

Other work suggests that WR-1065 could affect the functionality ofmolecular machines. For the purposes of illustration, only the molecularmachines known as origin recognition complexes will be discussed, butthe considerations presented below are applicable to many/all molecularmachines of viruses versus higher organisms. Because the components andstructure of these machines differ between simple life forms and higherlife forms, WR-1065-associated effects could result in differentconsequences. The basis for this hypothesis comes from two separateareas of consideration. The replicon model as proposed by Jacob et al.in 1964 has been shown to be applicable to all bacterial replicationsystems as well as to several viral families, including simian virus-40(SV-40). Bergsma et al. (1982) showed that initiation of replication inSV-40 required the interaction of a virally encoded initiator proteinwith a defined replicator sequence. In contrast to the study results,replication initiation-site selection within eukaryotic chromosomes isconsidered to be degenerate (Gilbert, 2004). For the latter chromosomes,multiple different replication initiation sites appear to exist, and nosingle obligatory site has been found.

Thus, it can be hypothesized that WR-1065 and similar compounds couldblock replication initiation through interactions with a definedreplicator sequence on viral nucleic acids, while not exerting a similareffect upon the nucleic acids of higher organisms because of thedegenerate nature of their initiation-site selection. Binding of WR-1065to replication initiation sites of eukaryotic cells would behypothesized to slow down nucleic acid processing and, hence, celldivision, but not block it completely. This hypothesis is supported bythe fact that WR-1065 has been shown to delay the progression of cellsthrough the cell cycle, but the exact mechanism remains unclear.

Second, replication origin recognition complexes of higher animals sharemany similarities with analogous complexes in simple life forms,including viruses and archaeal cells (Gai et al., 2004). However,critical differences have been noted in the components and in the modeof action of these molecular machines (Gai et al., 2004). To use SV-40as an example, the molecular machine known as the origin recognitioncomplex consists of the oncogenic large tumor antigen that is formed asa monomer; six monomers then form a hexamer, and two hexamers assembletogether, along with replication protein A, topoisomerase I, andpolymerase-alpha/primase. These molecules form the replicationinitiation complex for SV-40. The functionality of this complex ishighly dependent upon the conformation of individual monomers, as wellas the conformation of the hexamer (Gai et al. 2004). The functionalityof the entire replication initiation complex is also dependent upon theconformation of the target nucleic acids and the target initiation site(Gai et al., 2004). Because WR-1065 is known to have the capacity toalter the conformation of both nucleic acids and proteins, a complexsuch as that described for SV-40 would be especially vulnerable toinduced conformational changes. Partial support for this hypothesiscomes from studies demonstrating interactions between WR-1065 andtopoisomerase enzymes. Analogous molecular machines that operate incells of higher organisms would not be expected to share identicalconformational requirements.

In addition, because origin recognition complexes of eukaryotic cells donot appear to require an origin of replication site, but rather arecapable of using a variety of sites, it is reasonable to hypothesizethat these latter origin recognition complexes could potentiallytolerate a degree of conformational change that SV-40 and othervirally-associated origin recognition complexes cannot tolerate.

All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, as well asthe references of the Appendix 1, are incorporated herein by referencein their entirety and are hereby made a part of this specification. Tothe extent publications and patents or patent applications incorporatedby reference contradict the disclosure contained in the specification,the specification is intended to supersede and/or take precedence overany such contradictory material. The term “comprising” as used herein issynonymous with “including,” “containing,” or “characterized by,” and isinclusive or open-ended and does not exclude additional, unrecitedelements or method steps.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention.

1. A method of treating a virus infection in an individual in needthereof, comprising: administering to the individual an effectiveantiviral amount of a compound or a pharmaceutically acceptable salt orsolvate thereof, wherein the compound is of Formula (I) or Formula (II)

wherein X is selected from the group consisting of —PO₃H₂, hydrogen,acetyl, isobutyryl, pivaloyl, and benzoyl, wherein each of R₁, R₂, andR₃ is independently selected from hydrogen and C₁₋₆ alkyl, wherein n isan integer of from 1 to 10, and wherein the virus is a DNA virus, an RNAvirus, a DNA reverse transcribing virus or a non Retroviridae RNAreverse transcribing virus.
 2. The method of claim 1, wherein R₁ ismethyl, R₂ is hydrogen, R₃ is hydrogen, n is 3, and X is —PO₃H₂.
 3. Themethod of claim 1, wherein R₁ is methyl, R₂ is hydrogen, R₃ is hydrogen,n is 3, and X is hydrogen.
 4. The method of claim 1, wherein R₁ ishydrogen, R₂ is hydrogen, R₃ is hydrogen, n is 3, and X is —PO₃H₂. 5.The method of claim 1, wherein R₁ is hydrogen, R₂ is hydrogen, R₃ ishydrogen, n is 3, and X is hydrogen.
 6. The method of claim 1, whereinthe compound is administered to the individual at a daily dosage of fromabout 200 mg/m² to about 3000 mg/m².
 7. The method of claim 1, whereinthe step of administering is selected from the group consisting oforally administering, intravenously administering, parenterallyadministering, subcutaneously administering, and administering byinhalation.
 8. A method of treating a virus infection in an individualin need thereof, comprising: administering to the individual aneffective antiviral amount of: (i) amifostine, the free thiol form ofamifostine, the disulfide of amifostine (WR-33278), a combination ofboth the free thiol and the disulfide of amifostine, or otherstructurally and functionally related compounds; or (ii) phosphonol, thefree thiol form of phosphonol, the disulfide of phosphonol, acombination of both the free thiol and the disulfide of phosphonol, orother structurally and functionally related compounds, wherein the virusis a DNA virus, an RNA virus, a DNA reverse transcribing virus or a nonRetroviridae RNA reverse transcribing virus.
 9. (canceled)
 10. Themethod of claim 1, wherein said administering comprises administering tothe individual an effective antiviral amount of at least one compoundselected from the group consisting of:


11. The method of claim 1, wherein said administering comprisesadministering to the individual an effective antiviral amount of atleast one compound selected from the group consisting of:


12. A pharmaceutical kit comprising: a pharmaceutical compositioncomprising a compound or pharmaceutically acceptable salt or solvatethereof in a pharmaceutically acceptable carrier, the compound havingFormula (I) or Formula (II):

wherein X is selected from the group consisting of —PO₃H₂, hydrogen,acetyl, isobutyryl, pivaloyl, and benzoyl, wherein each of R₁, R₂, andR₃ is independently selected from hydrogen and C₁₋₆ alkyl, and wherein nis an integer of from 1 to 10; and directions for administering thepharmaceutical composition to a patient infected with a DNA virus, anRNA virus, a DNA reverse transcribing virus or a non Retroviridae RNAreverse transcribing virus.
 13. The method of claim 1, wherein the virusis a member of the Adenoviridae family or the Orthomyxoviridae family.